The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells

Abstract

T he development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigenpresenting cells, followed by endocytosis and presentation to naïve CD4 + T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4 + T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIII Y1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIII Y1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo. In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.

Tolerating Factor VIII: Recent Progress

“…These findings suggest that, at least in mice, the mannose receptor may not be directly involved in FVIII immunogenicity; the extent to which FVIII immunogenicity in humans depends on mannose receptors remains to be established. Apart from the mannoseending glycans linked to Asn 239 and Asn 2118 , exposed surface loops in the C-terminal FVIII C1 and C2 domains containing positively-charged residues have been implicated in the uptake of FVIII by both human (monocyte-derived) and murine (bone marrow-derived) dendritic cells (52,56,57). Modification of residues in the C1 domain surface loop containing Arg 2090 , Lys 2092 , and Met 2093 resulted in reduced FVIII inhibitor titers in FVIII-deficient mice (56).…”

Advances in knowledge of inhibitor formation in severe haemophilia A

“…Association with VWF protects FVIII from proteolysis and clearance from the plasma by shielding the C1 and C2 domains (Brinkhous et al , ; Fay et al , ). These domains of FVIII also play a role in the recognition of the protein by receptors on the surface of APCs, as several epitopes within the C1 and C2 domains have been identified as being necessary for APC binding and uptake (Dasgupta et al , ; Herczenik et al , ; Gangadharan et al , ). Secondly, in the presence of VWF, FVIII is slowly and inefficiently internalised by APCs.…”

Bioengineered molecules for the management of haemophilia: Promise and remaining challenges

“…However, with new insights on how FVIII interacts with antigen‐presenting cells, several approaches are being investigated. These include the expression of rFVIII within human cell lines with the aim of retaining human‐specific post‐translational modifications, such as its glycosylation profile; porcine‐human hybrids which eliminate highly immunogenic peptide sequences; Fc‐fusion and PEGylation; targeted mutagenesis of the FVIII C1 and C2 domains or fusion of FVIII with nanobodies with enhanced affinity for VWF, both of which aim to reduce dendritic cell endocytosis.…”